Assignment of interchain disulfide bonds in platelet-derived growth factor (PDGF) and evidence for agonist activity of monomeric PDGF.

Platelet-derived growth factor (PDGF) is a dimeric factor stabilized by disulfide bonds. Using an approach involving partial reduction of PDGF, we have identified the 2nd and 4th cysteine residues in the PDGF chains as the cysteine residues forming interchain disulfide bonds. Analysis of PDGF mutants in which the 2nd and 4th cysteine residues were mutated to serine residues revealed that the disulfide bonds are arranged in a cross-wise manner, with the 2nd cysteine residue in one chain being linked to the 4th cysteine residue in the other. A PDGF B-chain mutant, in which both the 2nd and 4th cysteine residues were substituted with serine residues, migrated as a monomer in sodium dodecyl sulfate gel electrophoresis and retained receptor binding activity. When analyzed in receptor dimerization and autophosphorylation assays, this mutant showed agonistic activity. Thus, structural information has been obtained that will allow the large scale production of properly folded monomeric PDGF, as well as design of specific PDGF heterodimers.     

Localization of the diphtheria toxin receptor-binding domain to the carboxyl-terminal Mr approximately 6000 region of the toxin

The carboxyl-terminal Mr = 5982 peptide of diphtheria toxin was prepared by specific cleavage of the toxin with hydroxylamine and purified by fast performance liquid chromatography. The identity of the peptide was established by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, reactivity with specific monoclonal antibodies, and amino-terminal sequence analysis. The Mr = 5982 peptide was shown to protect highly toxin-sensitive Vero cells from the lethal action of diphtheria toxin. This protection was shown to be due to inhibition of the initial step in the cytotoxic process, the binding of toxin to its receptor. These results strongly suggest that the Mr = 5982 carboxyl-terminal region (amino acid residues 482-535) is, or contains, the receptor-binding domain of diphtheria toxin.     

Analysis of the linkage positions in d-fructofuranosyl residues by the reductive-cleavage method

The suitability of the reductive-cleavage method for analysis of the linkage positions in d-fructofuranosyl residues of d-fructans was examined by using sucrose, chicory-root inulin, and Aerobacter levanicum levan as models. Permethylation, and reductive cleavage with triethylsilane in the presence of either boron trifluoride etherate or trimethylsilyl trifluoromethanesulfonate, gave the expected methylated derivatives of 2,5-anhydro-d-mannitol and 2,5-anhydro-d-glucitol. With either catalyst, nonreducing (terminal) d-fructofuranosyl groups and d-fructofuranosyl residues linked at O-1 gave derivatives having the manno configuration as the major product, whereas d-fructofuranosyl residues linked at O-6, and at both O-1 and O-6, gave derivatives having the gluco configuration as the major product. The independent synthesis, and n.m.r.- and mass-spectral characterization, of the methylated 2,5-anhydro-d-mannitol and 2,5-anhydro-d-glucitol derivatives formed from these residues by reductive cleavage are reported.     

Endotoxin and staphylococcus aureus enterotoxin a induce different patterns of cytokines

The effects of Staphylococcus aureus enterotoxin A (SEA) and lipopolysaccharide (LPS) in cytokine production were assessed at the single cell level in cells obtained from healthy blood donors. Cytokine production was studied with UV-microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies. The cytokines evaluated included tumour necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, IL-2, IL-4, interferon (IFN)-γ and TNF-β. LPS exhibited marked production of IL-1α, IL-1β, TNF-α, IL-6 and IL-8. After LPS stimulation IL-1α, IL-1β, TNF-α and IL-8 were the dominating products, all peaking at or before 4 hours after cell stimulation. In addition, IL-10 production was evident after 12 hours of cell stimulation. The T-lymphocyte-derived cytokines TNF-β, IL-2, IFN-γ and IL-4 were never detected in the cultures. All cytokine production, except IL-8, was downregulated at 96 hours.In contrast, peak production of IL-1α, IL-1β and IL-8, which were the dominant products, occurred after 12 hours in the SEA-stimulated cultures. Further, a significant T-lymphocyte production of TNF-β, TNF-α, IFN-γ and IL-2 was found with peak production 12–48 hours after initiation. Only low amounts of IL-6 were evident.The two types of cytokine pattern and kinetics found may correspond to the different clinical conditions after invasive Gram-negative Escherichia coli vs Gram-positive Staphylococcus aureus infections in humans, with a much more rapid onset of disease after E. coli infections. The data may also imply that different immunomodulating approaches should be considered in life-threatening diseases with the two microbacterial agents.     

Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts

We present a modified histochemical method for staining osteoclasts and adjacent mononuclear cells which takes advantage of the recently described substrate specificity for ATP of osteoclastic acid phosphatase. Staining of osteoclasts using ATP as substrate exhibits by light microscopy the same tartrate resistance as conventional acidic phosphatases, without the bone surface staining seen with other substrates. This feature, coupled with specific staining of fewer vicinal mononuclear cells, makes this method potentially useful for studying osteoclast ontogeny and function.     

Heat-shock protein 72 cell-surface expression on human lung carcinoma cells is associated with an increased sensitivity to lysis mediated by adherent natural killer cells

 The cell-surface expression patterns of major histocompatibility complex (MHC) class I, class II and heat-shock protein 72 (HSP72) molecules were measured on human lung (LX-1) and mammary (MX-1) carcinoma cells. No major differences were found in the MHC cell-surface expression pattern of both cell lines. However, they differ significantly in their capacity to express HSP72 on their cell surface. Under physiological conditions LX-1 cells express HSP72 molecules on more than 90% of the cells, whereas MX-1 cells exhibit no significant HSP72 cell-surface expression (less than 5%). These expression patterns remained stable in all further cell passages tested. The sensitivity to lysis mediated by an interleukin-2 (IL-2)-stimulated, adherent natural killer (NK) cell population could be correlated with the amount of cell-surface-expressed HSP72 molecules. By antibody-blocking studies, using HSP72-specific monoclonal antibody (mAb), a strong inhibition of lysis was only found with LX-1 cells but not with MX-1 cells. In contrast to the cell-surface expression, the cytoplasmic amount of HSP72 in MX-1 cells was twice as high compared to LX-1 cells under physiological conditions. After nonlethal heat-shock the rate of induction and the total cytoplasmic amounts of HSP72 were comparable in both cell lines. The clonogenic cell viability of LX-1 cells after incubation at temperatures ranging from 41°C to 44°C was significantly elevated compared to that of MX-1 cells. In conclusion we state the following: (i) HSP72 cell-surface expression on human carcinoma cells is independent of the cytoplasmic amount of HSP72; (ii) the cell-surface expression of HSP72 is associated with an increased sensitivity of tumour cells to lysis mediated by an IL-2-stimulated, adherent NK cell population; (iii) thermoresistance is not related to the cytoplasmic HSP72 level but might be related to the amount of HSP72 expressed on the cell surface. Received: 20 June 1996 / Accepted: 25 September 1996     

Ver?nderungen des Brutvogelbestandes im Gro?raum Bonn: Analyse der Rasterkartierung 1975 und 1985

Zusammenfassung Die Brutverbreitung der Vögel im Großraum Bonn wurde 1975 und 1985 in jeweils 221 Minutengitterfeldern von je 2,2 km² Größe kartiert. Statistisch signifikante Zunahmen besetzter Gitterfelder wurden für 9 Arten, Abnahmen für 22 Arten registriert, die z. T. auch andernorts festgestellt wurden. Unter Verwendung von Kartierungsdaten 1974–1978 konnte bei 14 Arten eine signifikante Änderung (Zunahmen: Sperber, Habicht, Singdrossel; Abnahmen: Steinschmätzer, Rebhuhn, Schafstelze, Grauammer, Mehlschwalbe, Feldsperling, Fasan, Dorngrasmücke, Hänfling, Feldlerche und Zaunkönig) nachgewiesen werden. Die Untersuchung zeigt, daß die Rasterkartierung gut geeignet ist, Aussagen über großflächige und langfristige Populationsveränderungen zu machen.

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Fluctuation of breeding populations in the area of Bonn: Analysis of grid mapping in 1975 and 1985

Summary The distribution of breeding birds in the area of Bonn was mapped in 1975 and 1985 in 221 grids of 2.2 km² size each. These data were analyzed to see whether fluctuations in the breeding populations occurred during the 11 years. It was based on the assumption that strong population changes should be reflected in the number of grids occupied by a species. Statistically significant increases were recorded for 9 species, decreases for 22 species. Taking other grid-map data from 1974 til 1978 (Wink 1980) into account, a continuous and significant trend could be established for 14 species (increase:Accipiter nisus, A. gentilis, Turdus philomelos; decrease:Oenanthe oenanthe, Perdix perdix, Motacilla flava, Emberiza calandra, Delichon urbica, Passer montanus, Phasianus colchicus, Sylvia communis, Acanthis cannabina, Alauda arvensis, Troglodytes troglodytes). This study shows that grid-mapping may provide valuable data on population trends of breeding birds.

     

Developmental stages of the hooded beetle Sericoderus lateralis (Coleoptera: Corylophidae) with comments on the phylogenetic position and effects of miniaturization

The first detailed morphological study of larvae, pupae and adults of a species of the hooded beetles (Coleoptera: Corylophidae) – Sericoderus lateralis – is presented. Histological sectioning, scanning and transmission electron microscopy, laser confocal microscopy and 3D-computer reconstruction were used. For the first time we report that according to the morphometric data of S. lateralis, at least some corylophid beetles have three larval stages. A phylogenetic position of Corylophidae within a cucujoid-cleroid clade is confirmed, and also the placement of Sericoderini within a corlyophid subgroup, which does not include Periptycinae and Foadiini. The larvae of Sericoderus are mainly characterized by plesiomorphic features compared to those of other corylophid tribes, notably Peltinodini and Rypobiini. Morphological and developmental consequences of miniaturization are discussed. Corylophid beetles display much less specific and far-reaching morphological consequences of miniaturization compared to Ptiliidae. We report the presence of unique modifications in the neural system not shared with any other insects, such as a distinctly asymmetric supraoesophageal ganglion in first instar larva, and a total displacement of the brain to the thorax in the adult stage. A highly unusual feature of the digestive tract is the sclerotised, V-shaped ventral wall of the pharynx. Developmental and size dependent changes in the relative volume of different organs are addressed. All organ systems change allometrically in the development of S. lateralis. Allometric trends in the volume of organs confirm that the factors limiting miniaturization are the size of the neural system, associated with the number and size of neurons (most critical for first instar larva), the mass of the skeleton, the egg size, and consequently the volume of the reproductive system (for free-living insects).